DNA recombination junction detection

ABSTRACT

The present invention provides methods, compositions and kits for detecting the presence or absence of an integrated insertion polynucleotide.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This patent application is a divisional of U.S. application Ser. No.11/803,102, filed May 11, 2007, now U.S. Pat. No. 8,187,812, whichclaims the benefit of U.S. Provisional Patent Application No.60/800,104, filed May 12, 2006, the entire disclosures of which arehereby incorporated herein by reference for all purposes.

BACKGROUND OF THE INVENTION

Detection of integrated insertion nucleotide sequences is important inmany contexts. For example, insertion nucleotide sequences can transportoncogenes in mammals, antibiotic resistance genes in bacteria and genesconveying identifiable traits in plants. Bacterial antibiotic resistanceis a major worldwide clinical problem and public health concern (see,for example, Sheldon, Clin Lab Sci (2005) 18:170 and French, Adv DrugDeliv Rev (2005) 57:1514). Clearly, an efficient, sensitive and reliablemethod for detecting the presence or absence of an integrated insertionnucleotide is valuable in clinical diagnostics and other contexts.

Others have developed methods for detecting integrated insertionnucleotide sequences. In one approach, the integrated insertionnucleotide is detected using PCR where one primer hybridizes to thetarget nucleic acid sequence and the other primer hybridizes to theinsertion nucleotide. Positive detection of the amplicon indicates thepresence of an insertion polynucleotide. This method can result in falsenegatives, or undetected insertion nucleotides, because the sequences ofinsertion nucleotides are often variable. Available primers may or maynot hybridize to the insertion polynucleotide. See, for example, theIDI-MRSAT™ Test by GeneOhm Sciences, San Diego, Calif.

In another approach, the integrated insertion nucleotide is detectedusing PCR where both primers hybridize to the insertion polynucleotide.Again, positive detection of amplicon indicates the presence of anintegrated insertion polynucleotide. This method also has theshortcoming that is can result in false negatives, due to thepolymorphic nature of integrated insertion nucleotides. Also, it is notclear whether the insertion polynucleotide is integrated into the targetnucleic acid sequence when both forward and reverse primers hybridize tothe insertion polynucleotide. See, for example, Kreiswirth, et al., JClin Microbiol (2005) 43:4585 and LightCycler® MRSA Detection Kit byRoche Diagnostics, Alameda, Calif.

In a further approach, presence or absence of the integrated insertionpolynucleotide is identified using PCR where one primer hybridizes tothe target nucleic acid sequence and the other primer hybridizes to asequence straddling the integration site between the target nucleic acidsequence and the insertion polynucleotide. Here, negative detection(lack of amplification) of amplicon indicates the presence of anintegrated insertion polynucleotide. This method has the disadvantagethat a negative signal indicates the positive integration of aninsertion polynucleotide (see, U.S. Pat. No. 6,156,507).

There remains a need for efficient, sensitive methods for detectingintegrated insertion polynucleotides which provide a positive signalindicative of the integration of the insertion polynucleotide.

BRIEF SUMMARY OF THE INVENTION

The present invention provides compositions (e.g., solutions, reactionmixtures), methods and kits for detecting the presence or absence of aninsertion polynucleotide in a nucleic acid. The compositions, methodsand kits find use in detecting, for example, integrated insertionpolynucleotides that confer resistance in bacteria to antibiotics.

With respect to the methods, the invention provides methods fordetermining the presence or absence of an integrated insertionpolynucleotide at a junction site in a target polynucleotide sequence.In some embodiments, the methods comprise contacting the targetpolynucleotide with a first primer, a second primer, a blockerpolynucleotide, a polymerase, and one or more nucleotide triphosphates,wherein:

(i) the target polynucleotide comprises a polynucleotide strandcomprising a junction site that, when an integrated insertionpolynucleotide is absent, is spanned on one side by a first targetsequence and on the other side by a second target sequence that iscontiguous with the first target sequence,

(ii) the blocker polynucleotide hybridizes to the contiguous first andsecond target sequences when the integrated insertion polynucleotide isabsent,

(iii) the first primer hybridizes to a first region of the first targetsequence that is proximal to the junction site, such that

(iv) the second primer hybridizes to a second region of the first targetsequence that is distal to the junction site, wherein the second primeris capable of priming synthesis of a copy of the first target sequencethat comprises the first and second regions of the first targetsequence,

such that when an integrated insertion sequence is present at thejunction site, the first and second primers support exponentialamplification of the first and second regions of the first targetsequence, and when an integrated insertion sequence is absent from thejunction site, the blocker polynucleotide hybridizes to the contiguousfirst and second target sequences so that amplification of the first andsecond regions of the first target sequence is inhibited, whereby thepresence or absence of the integrated insertion polynucleotide at thejunction site in the target polynucleotide is determined.

With respect to the compositions, the invention provides compositions,including reaction mixtures and solutions for determining the presenceor absence of an integrated insertion polynucleotide at a junction siteof a first target sequence and a second target sequence in a targetpolynucleotide. In some embodiments, the compositions comprise thetarget polynucleotide, a first primer, a second primer and a blockerpolynucleotide, wherein:

(i) the target polynucleotide comprises a polynucleotide strandcomprising a junction site that, when an integrated insertionpolynucleotide is absent, is spanned on one side by a first targetsequence and on the other side by a second target sequence that iscontiguous with the first target sequence,

(ii) the blocker polynucleotide hybridizes to the contiguous first andsecond target sequences when the integrated insertion polynucleotide isabsent,

(iii) the first primer hybridizes to a first region of the first targetsequence that is proximal to the junction site, such that

(iv) the second primer hybridizes to a second region of the first targetsequence that is distal to the junction site, wherein the second primeris capable of priming synthesis of a copy of the first target sequencethat comprises the first and second regions of the first targetsequence,

such that when an integrated insertion sequence is present at thejunction site, the first and second primers support exponentialamplification of the first and second regions of the first targetsequence, and when an integrated insertion sequence is absent from thejunction site, the blocker polynucleotide hybridizes to the contiguousfirst and second target sequences so that amplification of the first andsecond regions of the first target sequence is inhibited.

With respect to the kits, the invention provides kits for determiningthe presence or absence of an integrated insertion polynucleotide at ajunction site of a first target sequence and a second target sequence ina target polynucleotide. In some embodiments, the kits comprise a firstprimer, a second primer and a blocker polynucleotide, wherein:

(i) the target polynucleotide comprises a polynucleotide strandcomprising a junction site that, when an integrated insertionpolynucleotide is absent, is spanned on one side by a first targetsequence and on the other side by a second target sequence that iscontiguous with the first target sequence,

(ii) the blocker polynucleotide hybridizes to the contiguous first andsecond target sequences when the integrated insertion polynucleotide isabsent,

(iii) the first primer hybridizes to a first region of the first targetsequence that is proximal to the junction site, such that

(iv) the second primer hybridizes to a second region of the first targetsequence that is distal to the junction site, wherein the second primeris capable of priming synthesis of a copy of the first target sequencethat comprises the first and second regions of the first targetsequence,

such that when an integrated insertion sequence is present at thejunction site, the first and second primers support exponentialamplification of the first and second regions of the first targetsequence, and when an integrated insertion sequence is absent from thejunction site, the blocker polynucleotide hybridizes to the contiguousfirst and second target sequences so that amplification of the first andsecond regions of the first target sequence is inhibited.

With respect to further embodiments of the methods, compositions andkits, in some embodiments, the full length of the first primerhybridizes to the first target sequence competitively with the blocker.In some embodiments, a portion of the first primer hybridizes to thefirst target sequence competitively with the blocker.

In some embodiments, first region of the first target sequence isoutside the region of inverted repeats that can exist near a junction orintegration site. In some embodiments, first region of the first targetsequence is at least 20, 30, 40 or more nucleotide bases from thejunction site.

In some embodiments, the first primer and the blocker polynucleotideeach are substantially complementary to the first region within thetarget polynucleotide and the first primer has fewer mismatchednucleotides than the blocker polynucleotide relative to the first regionwithin the target polynucleotide. In some embodiments, the first primeris completely complementary to the first region within the targetpolynucleotide and the blocker polynucleotide carries at least oneinternal mismatch compared to the first region of the targetpolynucleotide.

In some embodiments, the first target sequence and the second targetsequence are portions of the Staphylococcus aureus orfX, the junctionsite is an attB integration site, the insertion polynucleotide is atleast a portion of a SCCmec complex, and the target polynucleotide isDNA from Staphylococcus aureus.

In some embodiments, the blocker is not extendable. In some embodiments,the blocker polynucleotide comprises a moiety at its 3′-end selectedfrom the group consisting of phosphate and hexylamine. In someembodiments, the blocker polynucleotide comprises at least one nucleicacid analog base.

In some embodiment, the nucleotide triphosphates are dNTP nucleotides.

In some embodiments, the polymerase is a DNA polymerase, for example, aTaq polymerase.

The methods, compositions and kits can be designed for the simultaneousevaluation of multiple target polynucleotides for the presence orabsence of an integrated insertion polynucleotide. In some embodiments,the methods are performed in a multiplex format.

In some embodiments, the methods further comprise the step of exposing,the reaction mixture or target polynucleotide to amplificationconditions. In some embodiments, the amplification is by PCR. In someembodiments, the methods are performed by multiplex PCR. In someembodiments, the amplification is detected by real-time PCR.

In some embodiments, the insertion polynucleotide is at least 10nucleotide bases in length. In some embodiments, the insertionpolynucleotide is integrated in the junction site. In some embodiments,the insertion polynucleotide is not integrated in the junction site.

In some embodiments, the kits further comprise a control targetpolynucleotide comprising the first target sequence and second targetsequence.

DEFINITIONS

An “insertion polynucleotide” or “insertion sequence” interchangeablyrefer to a polynucleotide that is not natively located at a particularinsertion site, but can be inserted within and become a contiguoussequence within the target polynucleotide sequence. The insertionpolynucleotide can originate from another place in the same sequence(e.g., transposons), or come from the genome of another organism (e.g.,a virus). An integrated insertion polynucleotide can be transmitted byan organism to its progeny. Non-limiting examples of insertionpolynucleotides include transposons, mobile genetic elements andretroviral vectors. Integration of the insertion polynucleotide can butneed not result in a detectable phenotype. For example, an insertionpolynucleotide can also include a nucleic acid sequence that confersantibiotic resistance in bacteria. An insertion polynucleotide can alsobe created by a DNA translocation event (e.g., a translocational tumormarker, including c-erb-B2 RNA splice variants; bcrlabl fusion; andtranslocations affecting Bcl-2 or Bcl-10) or can be any other markerjunction created by a DNA recombination event. See, FIGS. 1 and 2.

“Junction” or “junction site” interchangeably refer to a site at whichthe first target sequence abuts the second target sequence in a targetpolynucleotide sequence. A junction is also referred to as anintegration site. An insertion sequence is inserted into the targetpolynucleotide sequence at the junction or integration site. See, FIGS.1 and 2.

“Spanning the junction” refers to the ability of a polynucleotide tohybridize to two polynucleotides forming a junction by hybridizing tothe 5′-end of one polynucleotide and the 3′-end of the otherpolynucleotide. Polynucleotides spanning the junction of two sequenceshybridize to the 3′-end of the first sequence and the 5′-end of thesecond sequence, wherein the 3′ nucleotide of the first sequence and the5′ nucleotide of the second sequence are immediately adjacent to eachother.

The terms “proximal” or “adjacent” interchangeably refers to thepositioning of a region in the target polynucleotide sequencesubstantially complementary to and hybridizable to the first primer withrespect to the junction site in the target polynucleotide sequence. Theregion hybridizable to the first primer and junction site can beseparated by 1 to about 20 nucleotides, for example, about 1 to 10nucleotides. In some embodiments, the region hybridizable by the firstprimer and junction site directly abut one another.

“Amplification conditions” or “extension conditions” interchangeablyrefer to conditions under which a polymerase can add nucleotides to the3′ end of a polynucleotide. Such amplification or extension conditionsare well known in the art, and are described, for example, in Sambrookand Russell, Molecular Cloning: A Laboratory Manual, 3rd Edition, 2001,Cold Spring Harbor Laboratory Press and Ausubel, et al, CurrentProtocols in Molecular Biology, 1987-2007, John Wiley & Sons.

“Substantially complementary,” as used herein, refers to a sequencehaving no more than 20% (e.g., no more than 15, 10 or 5%) of thenucleotides in the sequence in question mismatched with a targetsequence. In some embodiments, the two polynucleotides have 1, 2, 3, 4,5, or more nucleotide mismatches.

A “mismatched nucleotide” refers to a nucleotide in a sequence ofinterest that is not the complement of the corresponding nucleotide in acorresponding sequence when the sequence of interest and the targetsequence are hybridized in an amplification reaction. The complement ofC is G and the complement of A is T. Those of skill in the art willappreciate that a variety of synthetic nucleotides which haveWatson-Crick binding properties are known and complementary syntheticnucleotides are intended to be encompassed by this definition.

A “blocker polynucleotide” refers to a polynucleotide that hybridizes toa target polynucleotide sequence that spans an integration site (i.e.,junction) for an insertion polynucleotide. In the absence of anintegrated insertion polynucleotide, a blocker polynucleotide hybridizesto both the first target sequence and the second target sequence of thetarget polynucleotide sequence. In some embodiments, the blockerpolynucleotide is not extendable by a polymerase.

As used herein, the terms “nucleic acid” and “polynucleotide” and arenot limited by length and are generic to linear polymers ofpolydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), and any other N-glycoside ofa purine or pyrimidine base, or modified purine or pyrimidine bases.These terms include double- and single-stranded DNA, as well as double-and single-stranded RNA.

A nucleic acid sequence or polynucleotide can comprise phosphodiesterlinkages or modified linkages including, but not limited tophosphotriester, phosphoramidate, siloxane, carbonate,carboxymethylester, acetamidate, carbamate, thioether, bridgedphosphoramidate, bridged methylene phosphonate, phosphorothioate,methylphosphonate, phosphorodithioate, bridged phosphorothioate orsulfone linkages, and combinations of such linkages.

A nucleic acid sequence or polynucleotide can comprise the fivebiologically occurring bases (adenine, guanine, thymine, cytosine anduracil) and/or bases other than the five biologically occurring bases.These bases may serve a number of purposes, e.g., to stabilize ordestabilize hybridization; to promote or inhibit probe degradation; oras attachment points for detectable moieties or quencher moieties. Forexample, a polynucleotide of the invention can contain one or moremodified, non-standard, or derivatized base moieties, including, but notlimited to, N⁶-methyl-adenine, N⁶-tert-butyl-benzyl-adenine, imidazole,substituted imidazoles, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxymethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil (i.e.,thymine), uracil-5-oxyacetic acidmethylester,3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine, and5-propynyl pyrimidine. Other examples of modified, non-standard, orderivatized base moieties may be found in U.S. Pat. Nos. 6,001,611;5,955,589; 5,844,106; 5,789,562; 5,750,343; 5,728,525; and 5,679,785,each of which is incorporated herein by reference in its entirety.

Furthermore, a nucleic acid sequence or polynucleotide can comprise oneor more modified sugar moieties including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and a hexose.

“Hybridization melting temperature” or “Tm” refers to the temperatureunder specified conditions at which a polynucleotide duplex is 50% insingle-stranded form and 50% in double-stranded form. Hybridizationmelting temperature is calculated using the nearest-neighbor two-statemodel, which is applicable to short DNA duplexes,Tm(° C.)=(ΔH°/(ΔS°+RIn[oligo]))−273.15where ΔH° (enthalpy) and ΔS° (entropy) are the melting parameterscalculated from the sequence and the published nearest neighborthermodynamic parameter, R is the ideal gas constant (1.987 cal K⁻¹mole⁻¹), [oligo] is the molar concentration of a polynucleotide, and theconstant of −273.15 converts temperature from Kelvin to degrees Celsius.Nearest neighbor parameters for DNA/DNA base pairs are obtained fromAllawi, et al., Biochemistry (1997) 36:10581; Allawi, et al.,Biochemistry (1998) 37:2170; Allawi, et al., Biochemishy (1998) 37:9435;and Peyret, et al., Biochemishy (1999) 38:3468. Tm depends on monovalentsalt concentration ([Na⁺]) of the solvent. The default concentration of[Na⁺] is 50 mM. Software programs for calculating Tm are readilyavailable, for example, from Integrated DNA Technologies, Coralville,Iowa.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A schematic showing the relationship between the first targetsequence, the second target sequence, the junction and the insertionpolynucleotide. The first and second target sequences can be from anon-coding sequence, the same or different reading frames or genesequence.

FIG. 2. A schematic showing the relationship between the hybridizationsites of the first primer, second primer, blocker polynucleotide,junction and insertion polynucleotide when the insertion sequence isabsent and when it is present. The blocker polynucleotide can containone or more internal mismatches with the target sequence.

FIG. 3. The results of an experiment demonstrating the effect of twodifferent blocker polynucleotides on the detection ofmethicillin-resistant Staphylococcus aureus (MRSA) andmethicillin-sensitive Staphylococcus aureus (MSSA) using the 5′-nucleaseassay. FAM end point fluorescence is indicated in relative fluorescenceunits (RFU).

FIG. 4. The results of an experiment demonstrating the effect of thepresence or absence of a blocker polynucleotide on the detection of MRSAand MSSA. FAM end point fluorescence is indicated in relativefluorescence units (RFU).

FIG. 5. The effect of Blocker GCG49 on 80 different clinical isolates ofMSSA. Data plotted as the change in threshold cycle (ΔCt;Ct_(with blocker)-Ct_(with blocker)), for the blocked MSSA strain,versus the Threshold cycle (Ct) of the unblocked MSSA strains.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods, compositions and kits fordetermining the presence or absence of an integrated insertionpolynucleotide. The invention allows for amplifying a nucleic acidsequence adjacent to an integrated insertion polynucleotide thereforeproviding a positive detection signal when the integrated insertionpolynucleotide is present and a negative detection signal when theintegrated insertion polynucleotide is absent.

The methods are directed to determining the presence of an integratedinsertion polynucleotide in a target polynucleotide sequence byamplifying a polynucleotide sequence adjacent to the integration site(i.e., junction). A positive amplification signal from primersindicating the presence of an integrated insertion polynucleotide isaccomplished by employing a blocker polynucleotide that hybridizes to atarget polynucleotide sequence spanning the junction in the absence ofan integrated insertion polynucleotide. See, FIG. 2. Preferably, theblocker can not be extended by a polymerase.

The blocker polynucleotide competes for hybridization to the targetpolynucleotide sequence with the first primer (i.e., “the competingprimer,” either forward or reverse) used to amplify the polynucleotidesequence adjacent to the integration site. In the absence of anintegrated insertion polynucleotide, the blocker polynucleotide has ahybridization melting temperature that is relatively higher than thehybridization melting temperature of the first (i.e., competitive)primer. Therefore, in the absence of an integrated insertionpolynucleotide, the blocker polynucleotide anneals to the targetpolynucleotide sequence and no polynucleotide sequence is amplified,indicating the absence of an integrated insertion polynucleotide. In thepresence of an integrated insertion polynucleotide, the blockerpolynucleotide has a hybridization melting temperature that isrelatively lower than the hybridization melting temperature of the first(i.e., competitive) primer. Therefore, in the presence of an integratedinsertion polynucleotide, the first (i.e., competing) primer anneals tothe target polynucleotide sequence, and an amplicon is amplified fromthe first (i.e., competitive) primer and the second (i.e.,non-competitive) primer, indicating the presence of an integratedinsertion polynucleotide. See, FIG. 2.

In some embodiments, the first primer and the blocker polynucleotidecompete for hybridization to a subsequence within the targetpolynucleotide sequence that is on the same side of the junction as thehybridization site of the second primer. Referring to FIG. 1, in thisembodiment, the first primer and the blocker polynucleotide compete forhybridization to a sequence within the first target sequence. The secondprimer also hybridizes to the first target sequence. In the presence ofan integrated insertion polynucleotide, the amplicon does not includethe sequence of the integrated insertion polynucleotide.

In some embodiments, the first primer and the blocker polynucleotidecompete for hybridization to a subsequence within the targetpolynucleotide sequence that is on the opposite side of the junctionfrom the hybridization site of the second primer. Referring to FIG. 1,in this embodiment, the first primer and the blocker polynucleotidecompete for hybridization to a sequence within the second targetsequence while the second primer hybridizes to the first targetsequence. In the presence of an integrated insertion polynucleotide, theamplicon includes the sequence of the integrated insertionpolynucleotide. In this embodiment, the insertion polynucleotidesequence is a length that can be practicably amplified, for example,less than about 2 kb.

Optionally, the relative hybridization Tm of the blocker polynucleotideand the first primer can be reversed such that amplification occurs whenthe integrated insertion polynucleotide is absent. In this case, thefirst primer and the blocker polynucleotide hybridize to a commonsequence in the first target sequence proximal to the junction, whereinthe blocker has a higher Tm for hybridizing to the common sequence thanthe first primer when the insertion polynucleotide is integrated intothe junction and the blocker polynucleotide has a lower Tm forhybridizing to the common sequence than the first primer when theintegrated insertion polynucleotide is not present in the junction,thereby allowing for amplification when the integrated insertion isabsent.

Methods

The methods find use in determining the presence or absence in a targetpolynucleotide sequence of an integrated insertion polynucleotide,including without limitation a mobile genetic element, a transposon, atranslocational fusion sequence (e.g., an oncogene, a tumor markerfusion sequence, a transgene insertion site, including a CRE/LOX site),or a retroviral genomic sequence.

Target Polynucleotide Sequence

The target polynucleotide sequence comprises a junction of a firsttarget sequence and a second target sequence, and optionally aninsertion polynucleotide (see, FIG. 1). In some embodiments, theinsertion polynucleotide is from the same genome as the targetpolynucleotide sequence (e.g., a transposon, a translocational fusionsequence). In some embodiments, the methods are used to determine thepresence or absence of a tumor marker translocation fusion sequence, forexample, c-erb-B2 RNA splice variants; bcr/abl fusion; andtranslocations affecting Bcl-2 or Bcl-10.

In some embodiments, the insertion polynucleotide is from a genomedifferent from the target polynucleotide sequence (e.g., a mobilegenetic element, a retroviral genomic sequence). The different genomecan be from the same or a different organism. In some embodiments, themethods are used to determine the presence or absence of a mobilegenetic element integrated into a bacterial host genome that conveysantibiotic resistance.

In some embodiments, the methods can be carried out for identifying thepresence or absence of an insertion polynucleotide integrated into atarget host genome. The host genome can be from any source. The genomicDNA can be prokaryotic or eukaryotic. It can be from bacteria, fungi,plants or animals. The host genomic DNA can be from a pathogen to aplant or animal host, for example, viral, bacterial, fungal orparasitic. Genomic DNA can be, for example, from bacterial generapathogenic to an animal host and capable of developing resistance toantimicrobial agents, including Staphylococcus, Streptococcus,Escherichia coli, Mycobacterium, Bacillus, Enterococcus, Enterobacter,Haemophilus, Pseudomonas, Klebsiella, Acinetobacter, Listeria,Helicobacter, Salmonella, Neisseria, Legionella, etc. In someembodiments, the host genomic DNA is from a Staphylococcus species, forexample, Staphylococcus aureus, Staphylococcus haemolyticus,Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcusxylosus, Staphylococcus warneri, Staphylococcus vitulinus,Staphylococcus sussinus, Staphylococcus simulans, Staphylococcus sciuri,etc.

The host genomic DNA can be from an animal host. The animal can be humanor non-human, and is can be mammalian, including domestic animals andagricultural animals. Domestic animals include canine, feline, rodent,lagomorpha, hamster, chinchilla, mutts, murine. Agricultural animalsinclude equine, bovine, ovine, porcine and chickens. The genomic DNA canbe tested from cells or tissues, as appropriate.

The host genomic DNA can be purified and/or isolated according totechniques well known in the art, for example, those described inSambrook and Russell, Molecular Cloning: A Lahoralory Manual, 3rdEdition, 2001, Cold Spring Harbor Laboratory Press and Ausubel, et al,Current Protocols in Molecular Biology, 1987-2007, John Wiley & Sons.

Insertion Polynucleotide

The insertion polynucleotide can be any nucleic acid sequence that canbe inserted between two linked polynucleotide sequences. The insertionpolynucleotide can, but need not, convey an identifiable phenotypictrait in the host when integrated into the target polynucleotidesequence. For example, as described above, the insertion polynucleotidecan be a retroviral genome sequence, a transposon, a translocationalfusion sequence or a mobile genetic element.

In some embodiments, the insertion polynucleotide is a mobile geneticelement that imparts resistance to antimicrobial agents. Mobile geneticelements that convey resistance to antibiotics in bacteria having themintegrated into their genomic DNA include those that encode sequencesthat neutralize the antibacterial mechanism of beta-lactam andaminoglycoside antibiotics.

For example, a mobile genetic element that encodes a penicillin bindingprotein can impart bacterial resistance to beta lactam antibiotics.Exemplified beta lactam antibiotics include methicillin, penicillins(e.g., penicillin G, penicillin V), amoxicillin, ampicillin, oxacillin,cloxacillin, dicloxacillin, nafcillin, carbenicillin, ticarcillin,mezlocillin, azlocillin, piperacillin, and the like. Beta lactamantibiotics are described, for example, in Chapter 45 of Goodman andGilman's Pharmacological Basis of Therapeutics, eds., Hardman andLimbird, 2001, McGraw-Hill. One example of a mobile genetic element thatconveys resistance to beta lactam antibiotics is the mecA complex, whichencodes a penicillin binding protein and imparts resistance tomethicillin, penicillins and other beta lactam antibiotics in bacteriawhen it is integrated into the bacterial host genome. ExemplifiedGenBank accession numbers for penicillin binding protein polypeptidesequences include YP_(—)252006; CAH17594, AAY60807; BAB07108; CAC95693;AAK39559; NP_(—)716793; and AAU27457. Exemplified GenBank accessionnumbers for nucleotide sequences encoding penicillin binding proteinpolypeptides include NC_(—)007168 (GeneID 3482097); AY894415; AP006716;AM048803; Y13096; EFY17797; EFA290435; and AE17323. Penicillin bindingproteins can be characterized by several common protein structuralmotifs, including MecA_N (pfam05223), PBP_dimer (pfam03717), andTranspeptidase (pfam00905).

A mobile genetic element that encodes an aminoglycosidephosphotransferase or an aminoglycoside acetyltransferase can impartbacterial resistance to aminoglycoside antibiotics. Exemplifiedaminoglycoside antibiotics include gentamicin, tobramcycin, amikacin,netilmicin, kanamycin, streptomycin and neomycin. Aminoglycosideantibiotics are described, for example, in Chapter 46 of Goodman andGilman's, supra. Exemplified GenBank accession numbers foraminoglycoside phospho- or acetyl-transferase polypeptide sequencesinclude YP_(—)253526; NP_(—)115315; CAD60196; AAC53691; AAX82584;AAK63041; AAT61777; AAG13458; AAK63040; and CAH19071. ExemplifiedGenBank accession numbers for nucleotide sequences encodingaminoglycoside phospho- or acetyl-transferase polypeptides includeNC_(—)007168 (GeneID 3482424). Aminoglycoside phospho- oracetyl-transferases can be characterized by common protein structuralmotifs, including APH (phosphotransferase enzyme family, pfam01636) andAcetyltransf_(—)1 (acetyltransferase (GNAT) family, pfam00583).

An insertion polynucleotide is at least about 10 nucleotide bases inlength, for example, about 10, 20, 50, 100, 500, 1000, 1500, 2000nucleotides bases in length, or longer.

Integration Sites

A target polynucleotide sequence susceptible to integration of aninsertion polynucleotide will have one or more integration sites. Theintegration site can be determined by the target polynucleotide sequenceor by the nature of the insertion polynucleotide. The methods aregenerally suited to detecting insertion sequences integrated atrecognition sites of site-specific recombinases (e.g., Cre, Flp orPhiC3l integrase), including without limitation loxP (e.g., loxP2,loxP3, loxP23, loxP511, loxB, loxC2, loxL, loxR), frt, dif, flp, and atttarget integration sequences. These are known in the art (see, forexample, Sorrell and Kolb, Biotechnol. Adv. (2005) 23:431; see also,Fluit and Schmitz, Clin Microbiol Infect (2004) 10:272).

In Staphylococcus, a site for integration (i.e., junction site) of theSCCmec complex carrying the mecA gene is the attB site within the orfXopen reading frame. Exemplified Staphylococcus orfX open reading framesequences include GenBank accession numbers NC_(—)007168 (GeneID3482010); NC_(—)002758 (GeneID 1119986); and NC_(—)007350 (GeneID3615252).

Primers

The primers of the invention are capable of acting as a point ofinitiation of DNA synthesis under conditions allowing for amplification,in which synthesis of a primer extension product complementary to anucleic acid strand is induced, i.e., in the presence of four differentnucleoside triphosphates and an agent for extension (e.g., a DNApolymerase or reverse transcriptase) in an appropriate buffer and at asuitable temperature (see, for example, Molecular Diagnostic PCRHandbook, Viljoen, et al., eds., 2005 Kluwer Academic Pub.; PCRProtocols, Bartlett, et al., eds., 2003, Humana Press; and PCR Primer: ALaboratory Manual, Dieffenbach, et al., eds., 2003, Cold Spring HarborLaboratory Press). A primer is preferably a single-stranded DNA. Theappropriate length of a primer depends on, for example, the intendedhybridization melting temperature (Tm) and location of the primer buttypically ranges from 10 to 50 nucleotides, preferably from 15-35 or18-22 nucleotides. Short primer molecules generally require lowertemperatures to form sufficiently stable hybrid complexes with thetemplate. A primer need not reflect the exact sequence of the templatenucleic acid, but must be sufficiently complementary to hybridize withthe template. In some embodiments, the primers of the invention can haveno mismatches, the same number of mismatches (e.g., 0, 1, 2, 3, 4, 5,etc.), or fewer hybridization mismatches in comparison to the blockerpolynucleotide. The primers of the invention can also have nohybridization mismatches. The design of suitable primers for theamplification of a given target sequence is well known in the art anddescribed in, for example, the literature cited herein.

The first (forward or reverse) primers of the invention compete with theblocker polynucleotide for hybridization to a common sequence within thetarget polynucleotide sequence (e.g., competitive displacement based onrelative Tm). The first primers are designed to have a relatively lowerhybridization melting temperature in comparison to the hybridizationmelting temperature of the blocker polynucleotide when no insertionnucleotide is present. The first primers further are designed to have arelatively higher hybridization melting temperature in comparison to thehybridization melting temperature of the blocker polynucleotide when aninsertion polynucleotide is present.

In some embodiments, the hybridization melting temperature of the first(i.e., competitive) primer will be between about 5° C.-15° C. higher orlower in comparison to the blocker polynucleotide, as appropriate. Insome embodiments, the hybridization melting temperature of the firstprimer is about 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 11° C., 12°C., 13° C., 14° C. or 15° C. higher or lower in comparison to theblocker polynucleotide, as appropriate. In some embodiments, thedifference in hybridization melting temperature can be more or less thanthis exemplified range.

The first and second primers of the invention can have a hybridizationmelting temperature ranging from about 50° C. to about 65° C., betweenabout 55° C. to about 60° C., for example. In some embodiments,hybridization melting temperature of a primer is about 50° C., 51° C.,52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C.,61° C., 62° C., 63° C., 64° C. or 65° C. However, the hybridizationmelting temperature of the first and second primers can be higher orlower than this temperature range.

Methods of designing a polynucleotide to have a desired hybridizationmelting temperature apply to both primers and blockers, and are known inthe art. Hybridization melting temperature can be adjusted, for example,by the number of guanosine (G) or cytidine (C) nucleosides, by adjustingthe length of the polynucleotide, or by incorporating one or more basesthat are mismatched with the target polynucleotide sequence. Generally,polynucleotides with greater numbers of G or C nucleosides, of longerlength, or with greater numbers of matching nucleosides will have higherhybridization melting temperatures. In some embodiments, thehybridization melting temperature differences between the first primerand the blocker are adjusted by introducing a greater number of basesmismatched with the target polynucleotide into the blocker sequence incomparison to the first primer sequence. For example, in someembodiments, the first primer has no bases mismatched with the targetpolynucleotide sequence and the blocker has one or more bases mismatchedwith the target polynucleotide sequence.

In some embodiments, the blocker polynucleotide and the first primerhybridize to a common sequence in the first target sequence proximal oradjacent to the junction that includes the full length of first primer.In some embodiments, the blocker polynucleotide and the first primerhybridize to a common sequence adjacent to the junction that iscomplementary to a portion of the first primer, for example 50%, 60%,70%, 80% or 90% of the 5′-end or 3′-end the of the first primer. Thecommon sequence competitively hybridized by blocker polynucleotide andthe first primer can be from 10-30 bases in length, for example, 12-25or 15-20 bases in length, for example, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 bases in length, orlonger or shorter.

The first primers are designed to anneal to a nucleic acid sequencewithin the target polynucleotide sequence that is proximal or adjacentto the integration site for the insertion polynucleotide. In theembodiments where the first primer and the blocker polynucleotidecompete to hybridize to a common sequence in the first target sequence,the 5′-end of the first primer can anneal within, e.g., about 30, 25 or20 nucleotide base positions of the integration site, for example,within about 15, 10 or 5 nucleotide base positions of the integrationsite, or sometimes abuts the integration site. In the embodiments wherethe first primer and the blocker polynucleotide compete to hybridize toa common sequence in the second target sequence, the 5′-end of the firstprimer can anneal within, e.g., about 40, 35 or 30 nucleotide basepositions of the integration site, for example, within about 25, 20 or15 nucleotide base positions of the integration site. The second primeranneals to a nucleic acid sequence in the target polynucleotide sequencesuch that the amplicon amplified from the first and second primer can bereliably detected. In some embodiments, the amplicon will be about 100,200, 300, 400, 500, 600, 800, 1000, 1500, 2000 nucleic acid bases inlength, or any integer of nucleic acid bases in length from about100-2000, but can be shorter or longer, as appropriate.

In some embodiments, the first and second primers anneal to an orfX genesequence in a Staphylococcus target polynucleotide sequence. In someembodiments, the first primer is a reverse primer comprising thesequence selected from the group consisting of5′-CTTATGATACGCTTCTCCTCGC-3′ (SEQ ID NO:2);5′-GCTTCTCCACGCATAATCTTAAATGCTCT-3′ (SEQ ID NO:9); and5′-TACTTATGATACGCTTCTCC-3′ (SEQ ID NO:10). In some embodiments, thesecond primer is a forward primer comprising the sequence5′-AGGGCAAAGCGACTTTGTATTC-3′ (SEQ ID NO: 1).

The primers can incorporate additional features which allow for thedetection or immobilization of the primer but do not alter the basicproperty of the primer, i.e., that of acting as a point of initiation ofDNA synthesis. For example, primers may contain an additional nucleicacid sequence at the 5′ end which does not hybridize to the targetpolynucleotide, but which facilitates cloning of the amplified product.The region of the primer which is sufficiently complementary to thetemplate to hybridize is referred to herein as the hybridizing region.In some embodiments, the first primer and/or the second primer compriseabout 1-10 consecutive cytosine or guanosine bases (SEQ ID NO:11) attheir 5′-end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutivecytosine or guanosine bases preceding the hybridizing region at the5′-end.

The first and second primers can also be modified and include at leastone nucleotide containing a sugar other than the conventional2′-deoxy-D-ribose or D-ribose found in naturally occurring DNA and RNA.Similarly, as used herein, a “modified polynucleotide” refers to apolynucleotide containing a sugar other than the conventional2′-deoxy-D-ribose or D-ribose found in naturally occurring DNA and RNA,and encompasses nucleotides in which the sugar is modified by theaddition or substitution of a side group, or in which the sugar is astereoisomer of the conventional 2′-deoxy-D-ribose or D-ribose found innaturally occurring DNA and RNA, or both. The terms are not used toindicate that a modified primer or nucleotide is the product of aprocess of modification, but rather to indicate the presence ofdifferences in the polynucleotide backbone relative to naturallyoccurring DNA or RNA. In particular, the primers of the presentinvention can be synthesized to contain a modified nucleotide, althoughthe chemical modification of a primer initially containing onlyconventional nucleotides can provide an alternative synthesis.

Blocker Polynucleotide

The blocking or blocker polynucleotides (“blockers”) of the invention donot operate as a point of extension under amplification or extensionconditions where the first and second primers operate as a point ofextension. In some embodiments, this is because they have a blockingmoiety attached to their 3′-end or because they lack a nucleophilicmoiety attached to their 3′-end (e.g., a hydroxyl moiety). A blockerpolynucleotide is preferably a single-stranded DNA. The appropriatelength of a blocker polynucleotide depends on, for example, the intendedhybridization melting temperature (Tm) and location of the blocker buttypically ranges from 25 to 60 nucleotides, preferably from about 30-50or 35-45 nucleotides, or any integer of nucleotide bases within theseranges. Shorter blocker polynucleotides generally require lowertemperatures to form sufficiently stable hybrid complexes with thetemplate. A blocker polynucleotide need not reflect the exact sequenceof the template nucleic acid, but must be sufficiently complementary tohybridize with the template. A blocker polynucleotide can hybridize to alonger or shorter sequence segment of the first target sequence relativeto the first primer. A blocker polynucleotide can be longer or shorterthan a first primer.

The Tm of the blocker polynucleotide can be varied by adjusting one ormore of several parameters, including for example, its length, thelocation and complementary sequence of the target polynucleotide towhich it hybridizes (i.e., hybridization condition), and the number ofinternal mismatches. The blocker polynucleotides of the invention canhave zero, one, two, three, four or more internal nucleotide basesmismatched with the target polynucleotide sequence, for example. Themismatched bases preferably are localized to the region of the blockerthat anneals to the first or second target sequence, i.e., thesubsequence in the target polynucleotide sequence in which the firstprimer and the blocker compete for hybridization. The design ofpolynucleotide sequences to hybridize to a target nucleotide sequence iswell known in the art and described in, for example, the literaturecited herein.

The blocker polynucleotide competes with the first primer of theinvention for hybridization to a common sequence within the first orsecond target subsequence within target polynucleotide sequence. See,FIGS. 1 and 2. The common target sequence competitively hybridized bythe blocker polynucleotide and the first primer can be complementary tothe full-length of the first primer or a portion of the first primer.The blocker polynucleotides are designed to have a relatively higherhybridization melting temperature in comparison to the hybridizationmelting temperature of the first primer when no insertion nucleotide ispresent. The blocker polynucleotides are further designed to have arelatively lower hybridization melting temperature in comparison to thehybridization melting temperature of the first primer when an insertionpolynucleotide is present. In some embodiments, the hybridizationmelting temperature of the blocker polynucleotide will be between about5° C.-15° C. higher or lower in comparison to the first primer, asappropriate. In some embodiments, the hybridization melting temperatureof the blocker polynucleotide is about 5° C., 6° C., 7° C., 8° C., 9°C., 10° C., 11° C., 12° C., 13° C., 14° C. or 15° C. higher or lower incomparison to the first primer, as appropriate. In some embodiments, thedifference in hybridization melting temperature can be more or less thanthis exemplified range.

For example, the blocker polynucleotides of the invention can have ahybridization melting temperature ranging from about 60° C. to about 75°C., for example, between about 65° C. to about 70° C., when no insertionnucleotide is present. In some embodiments, hybridization meltingtemperature of the blocker polynucleotides when no insertion nucleotideis present is about 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66°C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C. or75° C. The blocker polynucleotides can have a hybridization meltingtemperature ranging from about 40° C. to about 55° C., for example,between about 45° C. to about 50° C., when an insertion nucleotide ispresent. In some embodiments, hybridization melting temperature of theblocker polynucleotides when an insertion nucleotide is present is about40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C.,49° C., 50° C., 51° C., 52° C., 53° C., 54° C. or 55° C.

The blocker polynucleotides are designed to anneal to a nucleic acidsequence within the target polynucleotide sequence that overlaps theintegration site for the insertion polynucleotide. The blockerpolynucleotides can anneal about 30, 25, 20, 15 or 10 nucleotide basepositions spanning across the integration site or junction site.

In some embodiments, the blocker polynucleotide anneals to an orfX genesequence in a Staphylococcus host genome, straddling an attB integrationsite. In some embodiments, the blocker polynucleotide comprises one ormore polynucleotide sequences selected from the group consisting of:5′-CAGAATTTTTTAGTTTTACTTATGATACGCCTCTCCTCGC-3′ (SEQ ID NO:3);5′-TAAAAAACTCCTCCGCTACTTATGATACGCTTCTCCTCGC-3′ (SEQ ID NO:4);5′-CTCCTCATACAGAATTTTTTAGTTTTACTTATGATACGC CTCTCCTCGC-3′ (SEQ ID NO:6);5′-CTCCTCATACAGAATTTTTTAGTTTTACT TATGATACGCCTCTCCACGCATAATC-3′ (SEQ IDNO:7): and 5′-CTCCTCATACAGAATTTTTTAGTTTTACTTATGATACGCCTCTCCACGCATAATCTTAAATGC-3′ (SEQ ID NO:8).

Exemplified pairs of blocker polynucleotides and first primers (here,reverse primers) that hybridize to a common nucleic acid sequenceinclude those listed in Table 1, below.

TABLE 1 Blocker-GCG49 (SEQ ID NO: 6)CTCCTCATACAGAATTTTTTAGTTTTACTTATGATACGCCTCTCCTCGCReverse Primer (SEQ ID NO: 9) GCTTCTCCACGCATAATCTTAAATGCTCTBlocker-CTA55 (SEQ ID NO: 7)CTCCTCATACAGAATTTTTTAGTTTTACTTATGATACGCCTCTCCACGC ATAATCReverse Primer (SEQ ID NO: 9) GCTTCTCCACGCATAATCTTAAATGCTCTBlocker-CGT63 (SEQ ID NO: 8)CTCCTCATACAGAATTTTTTAGTTTTACTTATGATACGCCTCTCCACGC ATAATCTTAAATGCReverse Primer (SEQ ID NO: 9) GCTTCTCCACGCATAATCTTAAATGCTCT

The blocker polynucleotides preferably comprise a blocking moiety attheir 3′-end, that prevents their extension. Exemplified blockingmoieties include replacing the 3′-terminal hydroxyl group with ahydrogen, an amino or a phosphate group. In some embodiments, theblocking moiety on the blocker polynucleotide is a phosphate orhexylamine.

Modified Polynucleotides

Both primers and blocker polynucleotides will generally containphosphodiester bonds, although in some cases, as outlined herein,nucleic acid analogs can be used that may have alternate backbones,including, for example and without limitation, phosphoramide (Beaucageet al. (1993) Tetrahedron 49(10):1925 and references therein; Letsinger(1970) J. Org. Chem. 35:3800; Sprinzl et al. (1977) Eur. J. Biochem.81:579; Letsinger et al. (1986) Nucl. Acids Res. 14: 3487; Sawai et al.(1984) Chem. Lett. 805; Letsinger et al. (1988) J. Am. Chem. Soc.110:4470; and Pauwels et al. (1986) Chemica Scripta 26: 1419, which areeach incorporated by reference), phosphorothioate (Mag et al. (1991)Nucleic Acids Res. 19:1437; and U.S. Pat. No. 5,644,048, which are bothincorporated by reference), phosphorodithioate (Brio et al. (1989) J.Am. Chem. Soc. 111:2321, which is incorporated by reference),O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press (1992), whichis incorporated by reference); and peptide nucleic acid (PNA) backbonesand locked nucleic acid backbones (LNA) and linkages (see, Egholm (1992)J. Am. Chem. Soc. 114:1895; Meier et al. (1992) Chem. Int. Ed. Engl.31:1008; Nielsen (1993) Nature 365:566; and Carlsson et al. (1996)Nature 380:207, Demidov, Trends Biotechnol (2003) 21:4-7; and Vester andWengel, Biochemistry (2004) 43:132:33-41, which are each incorporated byreference).

Other analog nucleic acids include those with positively chargedbackbones (Denpcy et al. (1995) Proc. Natl. Acad. Sci. USA 92:6097,which is incorporated by reference); non-ionic backbones (U.S. Pat. Nos.5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew (1991)Chem. Intl. Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem.Soc. 110:4470; Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597;Chapters 2 and 3, ASC Symposium Series 580, Ed. Y. S. Sanghvi and P. DanCook; Mesmaeker et al. (1994) Bioorganic & Medicinal Chem. Lett. 4: 395;Jeffs et al. (1994) J. Biomolecular NMR 34:17; and Tetrahedron Lett.37:743 (1996), which are each incorporated by reference) and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Ed. Y. S.Sanghvi and P. Dan Cook, which references are each incorporated byreference. Nucleic acids containing one or more carbocyclic sugars arealso included within the definition of nucleic acids (see Jenkins et al.(1995) Chem. Soc. Rev. pp 169-176, which is incorporated by reference).Several nucleic acid analogs are also described in, e.g., Rawls, C & ENews Jun. 2, 1997 page 35, which is incorporated by reference. Thesemodifications of the ribose-phosphate backbone can be used to facilitatethe addition of additional moieties such as labels, or to alter thestability and half-life of such molecules in physiological environments.

In addition to the naturally occurring heterocyclic bases that aretypically found in nucleic acids (e.g., adenine, guanine, thymine,cytosine, and uracil), nucleic acid analogs also include those havingnon-naturally occurring heterocyclic or modified bases, many of whichare described, or otherwise referred to, herein. In particular, manynon-naturally occurring bases are described further in, e.g., Seela etal. (1991) Helv. Chico. Acta 74:1790, Grein et al. (1994) Bioorg. Med.Chem. Lett. 4:971-976, and Seela et al. (1999) Helv. Chim. Acta 82:1640,which are each incorporated by reference. To further illustrate, certainbases used in nucleotides that act as melting temperature (Tm) modifiersare optionally included. For example, some of these include7-deazapurines (e.g., 7-deazaguanine, 7-deazaadenine, etc.),pyrazolo[3,4-d]pyrimidines, propynyl-dN (e.g., propynyl-dU, propynyl-dC,etc.), and the like. See, e.g., U.S. Pat. No. 5,990,303, which isincorporated by reference. Other representative heterocyclic basesinclude, e.g., hypoxanthine, inosine, xanthine; 8-aza derivatives of2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine,inosine and xanthine; 7-deaza-8-aza derivatives of adenine, guanine,2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine,inosine and xanthine; 6-azacytosine; 5-fluorocytosine; 5-chlorocytosine;5-iodocytosine; 5-bromocytosine; 5-methylcytosine; 5-propynylcytosine;5-bromovinyluracil; 5-fluorouracil; 5-chlorouracil; 5-iodouracil;5-bromouracil; 5-trifluoromethyluracil; 5-methoxymethyluracil;5-ethynyluracil; 5-propynyluracil, and the like. Examples of modifiedbases and nucleotides are also described in, e.g., U.S. Pat. Nos.5,484,908, 5,645,985, 5,830,653, 6,639,059, 6,303,315 and U.S. Pat.Application Pub. No. 2003/0092905, which are each incorporated byreference.

Amplification Reactions

Amplification of an RNA or DNA template using reactions is well known(see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide toMethods and Applications (Innis et al., eds, 1990)). Methods such aspolymerase chain reaction (PCR) and ligase chain reaction (LCR) can beused to amplify nucleic acid sequences of target DNA sequences directlyfrom mRNA, from cDNA, from genomic libraries or cDNA libraries. Thereaction is preferably carried out in a thermal cycler to facilitateincubation times at desired temperatures. See, e.g., PCR PRIMER, ALABORATORY MANUAL (Dieffenbach, ed. 2003) Cold Spring Harbor Press.

Exemplary PCR reaction conditions allowing for amplification of anamplicon typically comprise either two or three step cycles. Two stepcycles have a denaturation step followed by a hybridization/elongationstep. Three step cycles comprise a denaturation step followed by ahybridization step followed by a separate elongation step.

In some embodiments, an amplified amplicon in the presence of aninsertion polynucleotide integrated into the target polynucleotide isdetected by real-time PCR. Real-time RT-PCR is a method that utilizesspecifically engineered DNA sequences (two primers and a fluorescentlylabeled probe) to detect and quantify target sequences of DNA. The probecontains a fluorescent reporter dye on one end and a quencher dye on theother. During each amplification cycle, the probe first attaches to thetarget sequence of DNA, followed by attachment of the primers. As theDNA strand is copied, the reporter dye is released from the probe andemits a fluorescent signal. The amount of fluorescence increases witheach cycle of PCR in proportion to the amount of target DNA. Thisresults in direct detection and quantification of the target DNAsequence with a high degree of specificity, accuracy, and sensitivity.

In some embodiments, the multiple amplification reactions are performedby multiplex PCR. Multiplex PCR reactions refer to a PCR reaction wheremore than one primer set is included in the reaction pool allowing 2 ormore different. DNA targets to be amplified by PCR in a single reactiontube. Multiplex PCR can be quantitative and can be evaluated“real-time.” Multiplex PCR reactions are useful for validation,diagnostic and prognostic purposes. Multiplex PCR reactions can becarried out using manual or automatic thermal cycling. Any commerciallyavailable thermal cycler may be used, such as, e.g., Perkin-Elmer 9600cycler. Using multiplex PCR, at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,50, 100 or more target polynucleotides can be evaluated for the presenceor absence of an integrated insertion polynucleotide.

Isothermic amplification reactions are also known and can be usedaccording to the methods of the invention. Examples of isothermicamplification reactions include strand displacement amplification (SDA)(Walker, et al. Nucleic Acids Res. 20(7): 1691-6 (1992); Walker PCRMethods Appl 3(1):1-6 (1993)), transcription-mediated amplification(Phyffer, et al., J. Clin. Microbiol. 34:834-841 (1996); Vuorinen, etal., J. Clin. Microbiol. 33:1856-1859 (1995)), nucleic acidsequence-based amplification (NASBA) (Compton, Nature 350(6313):91-2(1991), rolling circle amplification (RCA) (Lisby, Mol. Biotechnol.12(1):75-99 (1999)); Hatch et al., Genet. Anal. 15(2):35-40 (1999)) andbranched DNA signal amplification (bDNA) (see, e.g., Iqbal et al., Mol.Cell Probes 13(4):315-320 (1999)). Other amplification methods known tothose of skill in the art include CPR (Cycling Probe Reaction), SSR(Self-Sustained Sequence Replication), SOA (Strand DisplacementAmplification), QBR (Q-Beta Replicase), Re-AMP (formerly RAMP), RCR(Repair Chain Reaction), TAS (Transorbtion Based Amplification System),and HCS.

The concentration of the magnesium salt in the reaction mixture can beimportant when trying to copy different target DNA sequences. Thus, somevariation of the concentration of the magnesium salt, e.g., magnesiumchloride, may be required to optimize the reaction to amplify the targetpolynucleotide sequences of interest. One of skill can vary theconcentration of magnesium salt or ion present in the reaction mixtureto arrive at the proper conditions for amplification.

In some embodiments, a second pair of primers is included in theamplification reaction that allow for the amplification of a controlsequence in the target polynucleotide, for example, to quantify theamount of target polynucleotide in an assay.

In some embodiments, the molar ratio of the one or more blockerpolynucleotides to the competitive first primer in the amplificationreaction can be optimized. In some embodiments, the molar ratio of theone or more blocker polynucleotides to the competitive first primer inthe amplification reaction is greater than a 1:1 molar ratio of blockerpolynucleotide(s) to first primer, for example, from about 5:1 to about30:1, for example, about a 5:1, 10:1, 20:1, 25:1 or 30:1 molar ratio ofblocker polynucleotide(s) to first primer.

Detection of Amplified Polynucleotides

Amplified nucleic acid sequences (“amplicons”) can be detected using anymethod known in the art. For example, amplicons can be detected in anagarose gel using ethidium bromide. The presence of an insertionpolynucleotide is indicated by the presence of an amplicon signal or theincreased presence of an amplicon signal in comparison to a controltarget polynucleotide sequence, for example, a target polynucleotidesequence known not to have an insertion polynucleotide, for example, amethicillin-sensitive Staphylococcus aureus (MSSA).

In some embodiments, amplicons are detected using probes thatspecifically hybridize to the amplicon and are detectable uponhybrizidation to the amplicon. Numerous types of probes are capable ofhybridizing to and detecting a particular polynucleotide sequences. Insome cases, the probe also comprises a fluorophore or enzyme, asdescribed below, which allows for the detection of the binding of theprobe to its complementary target sequence.

Probe concentration should be sufficient to bind to the amount of targetor control sequences that are amplified so as to provide an accurateassessment of the quantity of amplified sequence. Those of skill in theart will recognize that the amount of concentration of probe will varyaccording to the binding affinity of the probe as well as the quantityof sequence to be bound. Typical probe concentrations will range from0.01 μM to 0.5 μM. Typical probe length will range from about 20-40nucleotide bases in length, for example, about 25-35 nucleotide bases inlength, or any integer number of nucleotide bases within these ranges.

The present invention can employ many different kinds of nucleic acidhybridization probes for detection of amplicon. Typically, for signalgeneration, the probes utilize a change in the fluorescence of afluorophore due to a change in its interaction with another molecule ormoiety brought about by changing the distance between the fluorophoreand the interacting molecule or moiety. Alternatively, amplicons can bedetected using other methods, for example, using radioactively-labeledor enzymatically-labeled probes.

In some instances, multiple fluorescent labels are employed. In apreferred embodiment, at least two fluorescent labels are used which aremembers of a fluorescence resonance energy transfer (FRET) pair. FRET isphenomenon known in the art wherein excitation of one fluorescent dye istransferred to another without emission of a photon. A FRET pairconsists of a donor fluorophore and an acceptor fluorophore. Thefluorescence emission spectrum of the donor and the fluorescenceabsorption spectrum of the acceptor usually overlap, and the twomolecules must be in close proximity. The distance between donor andacceptor at which 50% of donors are deactivated (transfer energy to theacceptor) is defined by the Forster radius (R_(o)), which is typically10-100 Å. Changes in the fluorescence emission spectrum comprising FRETpairs can be detected, indicating that they are in close proximity(i.e., within 100 Å of each other). This will typically result from thebinding or dissociation of two molecules, one of which is labeled with aFRET donor and the other of which is labeled with a FRET acceptor,wherein such binding brings the FRET pair in close proximity. Binding ofsuch molecules will result in an increased fluorescence emission of theacceptor and/or quenching of the fluorescence emission of the donor.

FRET pairs (donor/acceptor) useful in the invention include, but are notlimited to, EDANS/fluorescein, IAEDANS/fluorescein,fluorescein/tetramethylrhodamine, fluorescein/LC Red 640, fluorescein/Cy5, fluorescein/Cy 5.5 and fluorescein/LC Red 705.

in another aspect of FRET, a fluorescent donor molecule and anonfluorescent acceptor molecule (“quencher”) may be employed. In thisapplication, fluorescent emission of the donor will increase whenquencher is displaced from close proximity to the donor and fluorescentemission will decrease when the quencher is brought into close proximityto the donor. Useful quenchers include, but are not limited to, DABCYL,QSY 7 and QSY 33. Useful fluorescent donor/quencher pairs include, butare not limited to EDANS/DABCYL, Texas Red/DABCYL, BODIPY/DABCYL,Lucifer yellow/DABCYL, coumarin/DABCYL and fluorescein/QSY 7 dye.

The skilled artisan will appreciate that FRET and fluorescence quenchingallow for monitoring of binding of labeled molecules over time,providing cycle-dependent information regarding the time course ofbinding reactions.

In some embodiments, the amplified nucleic acid sequence is detectedusing a probe labeled at its 5′-end with a fluorophore and at its 3′-endwith a quencher. In a further embodiment, the fluorophore is fluorescein(FAM) and the quencher is QSY7. Alternatively, fluorophore(s) and/orquencher(s) can be located at an internal site within a probe.

In some embodiments, the detectable probe hybridizes to an ampliconcorresponding to a Staphylococcus orfX sequence. In a furtherembodiment, the detectable probe comprises the sequence5′-CGGCCTGCACAAGGACGTCTTACAACGTAG-3′ (SEQ ID NO:5).

Another type of nucleic acid hybridization probe assay utilizing a FRETpair is the “TaqMan®” assay described in Gelfand et al. U.S. Pat. No.5,210,015, and Livak et al. U.S. Pat. No. 5,538,848. The probe is asingle-stranded polynucleotide labeled with a FRET pair. In a TaqMan®assay, a DNA polymerase releases single or multiple nucleotides bycleavage of the polynucleotide probe when it is hybridized to a targetstrand. That release provides a way to separate the quencher label andthe fluorophore label of the FRET pair.

Yet another type of nucleic acid hybridization probe assay utilizingFRET pairs is described in Tyagi et al. U.S. Pat. No. 5,925,517, whichutilizes labeled polynucleotide probes, which are referred to as“Molecular Beacons.” See Tyagi, S, and Kramer, F. R., NatureBiotechnology 14: 303-308 (1996). A molecular beacon probe is apolynucleotide whose end regions hybridize with one another to form ahairpin in the absence of target but are separated if the centralportion of the probe hybridizes to its target sequence. When the probehybridizes to a target, that is, when the target-recognition sequencehybridizes to a complementary target sequence, a relatively rigid helixis formed, causing the stem hybrid to unwind and forcing the arms apart.

Non-FRET fluorescent probes can also be used. For example, changes inthe absorption spectra of the label pair can be used as a detectablesignal as an alternative to change in fluorescence. When change inabsorption is utilized, the label pair may include any two chromophores,that is, fluorophores, quenchers and other chromophores. The label pairmay even be identical chromophores.

Compositions

The invention further provides compositions, including for example,solutions, reaction mixtures, and reaction vessels comprising the firstand second primers, blocker polynucleotides and optionally detectionprobes, as described above.

In some embodiments, compositions comprise first and second primers, anda blocker polynucleotide that each hybridize to a Staphylococcus orfXsequence, wherein the first primer and the blocker polynucleotidecompete with each other to hybridize to a common subsequence adjacent toan attB integration site within the orfX sequence. Hybridization of theblocker polynucleotide is favored when the insertion polynucleotide isnot present, and hybridization of the first primer is favored when theinsertion polynucleotide is present (e.g., a SCCmec complex comprising amecA gene).

In some embodiments, the compositions include a plurality of first andsecond primers and/or a plurality of blocker polynucleotides, forexample, for performing multiplex PCR.

The compositions can further include a target polynucleotide sequence(e.g., a genomic nucleic acid sequence from a host cell), nucleotidebases (dNTPs including dATP, dCTP, dGTP, dTTP, dUTP), polymerases, andappropriate reaction buffers, salts and metal ions. In some embodiments,the polymerase is a Taq polymerase, although any thermostable polymeraseor DNA polymerase suitable for nucleotide sequence amplification or DNAextension reactions can be included. Thermostable polymerases are wellknown in the art and are readily commercially available (for example,from New England Biolabs, Ipswich, Mass.; Promega, Madison, Wis.;Stratagene, La Jolla, Calif.; Roche Applied Science, Indianapolis,Ind.). In some embodiments, the compositions further include one or moredetection probes for detecting amplification of an amplicon, forexample, for real-time amplification detection.

Kits

The invention further provides kits comprising the first and secondprimers, one or more blocker polynucleotides and optionally detectionprobes, as described above for the compositions and methods. The kitsoptionally can further comprise a control target polynucleotide, forexample, a control genomic sequence from a host cell known to haveintegrated or known not to have integrated an insertion polynucleotide.In addition, the kit can include amplification reagents, includingnucleotides (e.g., A, C, G and T), a DNA polymerase and appropriatebuffers, salts and other reagents to facilitate amplification reactions.In some embodiments, the kits further include one or more detectionprobes for detecting amplification of an amplicon, for example, forreal-time amplification detection. In some embodiments, the kits includea plurality of first and second primers and/or a plurality of blockerpolynucleotides, for example, for performing multiplex PCR. The kits canalso include written instructions for the use of the kit to amplify andcontrol for amplification of a target sample.

In some embodiments, the invention provides kits that include one ormore reaction vessels that have aliquots of some or all of the reactioncomponents of the invention in them. Aliquots can be in liquid or driedform. Reaction vessels can include sample processing cartridges or othervessels that allow for the containment, processing and/or amplificationof Samples in the same vessel. In some embodiment, the kits furthercomprise a multiwell substrate (e.g., multivessel strip, multivesselplate), for example, for concurrently amplifying from a plurality oftarget polynucleotides. Such kits allow for ready detection ofamplification products of the invention into standard or portableamplification devices.

In some embodiments, the kits comprise vessels such as sample processingcartridges useful for rapid amplification of a sample as described inBelgrader, P., et al., Biosensors and Bioelectronics 14:849-852 (2000);Belgrader, P., et al., Science, 284:449-450 (1999); and Northrup, M. A.,et al. “A New Generation of PCR Instruments and Nucleic AcidConcentration Systems” in PCR PROTOCOLS (Sninsky, J. J. et al (eds.))Academic, San Diego, Chapter 8 (1998)).

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Detection of Methicillin-Resistant Staphylococcus aureus(MRSA) Using an SCCmec Junction Blocker Polynucleotide

Real-time PCR (RT-PCR; 5′-nuclease assay) reactions were carried out todetermine the presence or absence of an SCCmec integrated into thegenomic DNA of MRSA and MSSA isolates at the insertion site. Thereaction used two primers that hybridized to orfX sequences, afluorescently-labeled probe and one of two blocker polynucleotidesdesigned to accommodate sequence polymorphisms known to occur in theorfX region of S. aureus (below).

orfX forward primer (F1): (SEQ ID NO: 1) 5′-AGGGCAAAGCGACTTTGTATTC-3′orfX reverse primer (R11): (SEQ ID NO: 2) 5′-CTTATGATACGCTTCTCCTCGC-3′

Separate reactions were carried out in the presence of each of thefollowing blocker polynucleotide sequences:

(SEQ ID NO: 12) 5′-CAGAATTTTTTAGTTTTACTTATGATACGCCTCTCCTCGC-PO₃-3′.(SEQ ID NO: 13) 5′-TAAAAAACTCCTCCGCTACTTATGATACGCTTCTCCTCGC-PO₃-3′.The results are shown in Table 2 and in FIG. 3.

TABLE 2 S. aureus FAM End point FAM Threshold Protocol strainfluorescence (RFU) Cycle (C₁) Blocker 3 MRSA 143.71 37.13 MRSA 138.9537.39 MSSA 11.65 ND* MSSA 8.59 ND* Blocker 4 MRSA 150.79 36.96 MRSA147.83 36.89 MSSA 10.52 N.D*. MSSA 9.64 N.D*. *ND = C₁ not detected. Thefluorescent signal did not cross the threshold during 45 cycles ofreal-time PCR indicating that the orfX amplicon was not detected.

As can be seen from the Table, a subsequence of the orfX region wasamplified from MSSA strain genomic DNA in the absence of the blockerpolynucleotide. However, the orfX subsequence was not amplified in wheneither of the two blocker polynucleotides were present in the reaction.A target within the orfX region was amplified and detected with a robustpositive signal from MRSA genomic DNA in the presence or the absence ofeither blocker polynucleotide.

Example 2 Detection of orfX from MRSA and MSSA Genomic DNA in thePresence or Absence of an SCCmec Junction Blocker Polynucleotide

An RT-PCR was carried out to determine the effect of a blockerpolynucleotide on the amplification of an orfX amplicon from the hostgenomes of MRSA and MSSA strains. PCR reactions were carried out usingforward and reverse primers that hybridized to orfX and theamplification was detected with a FAM-labeled probe (below).

orfX forward primer (F1): (SEQ ID NO: 1) 5′-AGGGCAAAGCGACTTTGTATTC-3′orfX reverse primer (R11): (SEQ ID NO: 2) 5′-CTTATGATACGCTTCTCCTCGC-3′orfX probe: (SEQ ID NO: 14)5′-FAM-CGGCCTGCACAAGGACGTCTTACAACGTAG-Quencher 3′

The results are shown in Table 3 and in FIG. 4.

TABLE 3 S. aureus FAM End point FAM Threshold strain BlockerFluorescence (RFU) Cycle (C₁) MRSA − 478.39 25.74 MRSA − 517.99 25.55MRSA + 245.12 36.23 MRSA + 220.29 36.58 MSSA − 507.85 26.93 MSSA −493.96 26.93 MSSA + 4.19 ND* MSSA + 1.72 ND* *ND = C₁ not detected. Thefluorescent signal did not cross the threshold within 45 cycles ofreal-time PCR indicating that the orfX amplicon was not detected.

As can be seen from Table 3, the orfX subsequence was not amplified fromMSSA genomic DNA in the presence of the blocker polynucleotide. In theabsence of the blocker, an orfX subsequence was amplified. The orfXsubsequence was amplified with a robust positive signal from MRSAgenomic DNA in the presence and absence of a blocker polynucleotide.

Example 3 Testing of Blocker GCG49

In this example, blocker GCG49 was tested for its ability to blockamplification of target polynucleotide sequences (orfX) in 80 differentclinical isolates of MSSA. The sequences of the orfX and attB regions ofthese 80 isolates were not known, but these isolates were chosen torepresent a cross-section of S. aureus strains that might be encounteredin the clinical environment.

The nucleotide sequence of MSSA strain ATCC 35556 in the orfX region isshown in Table 4, along with the blocker, primers and probe sequences.

TABLE 4 MSSA Target, Blocker GCG49 and PCR Primer Sequences

Blocker GCG49 (highlighted)CTCCTCATACAGAATTTTTTAGTTTTACTTATGATACGCCTCTCCTCGC  (SEQ ID NO: 6)Probe MI80 (italics; mismatches in bold)ccccGCTTCTCCACGCATAATCTTAAATGCTCT (SEQ ID NO: 16)Primer R6 (double underline) TACTTATGATACGCTTCTCC (SEQ ID NO: 10)Primer (single underline)CAATTAACACAACCCGCATCATTTGATGTGGG (SEQ ID NO: 17)Procedure:

Crude extracts of DNA from 80 isolates of MSSA were obtained by firstpure-streaking these bacterial strains in Petri plates contain an agarmedium. A single, isolated colony from a given strain was picked up withan inoculating loop, and then suspended in one milliliter of molecularbiology-grade water. This process was repeated for all 80 isolates. Thetubes were capped, then heated to 95° C. for 10-15 minutes. After thesolutions were cooled, these DNA extracts were centrifuged for at10,000×g for 5 minutes to remove cellular debris. A several microliteraliquot of the DNA-containing supernatant was diluted 100-fold in a 10mM Tris buffer (pH 8.3) containing 1 mM EDTA. This diluted aliquot oflysate was then used as the test DNA sample. The amount of DNA presentin these samples was not quantified. Because of the simple method usedfor preparation the DNA quantity was certain to be varied frompreparation to preparation. The DNA samples were then subject toreal-time, quantitative PCR with or without the presence of BlockerGCG49.

The results are depicted in FIGS. 5 and 6. In the absence of blockerpolynucleotide GCG49 OrfX was amplified from the genomic DNA of all 80tested MSSA strains. The Ct value observed for these RT-PCRs isproportional to the amount of DNA target present at the start of thereaction. The Ct value will be lower when a greater quantity of targetDNA is present, and the C_(t) will be higher when lower quantities ofDNA are present, i.e. when higher quantities of DNA are present, thefluorescence will cross the detection threshold in fewer cycles thanwhen a lesser amount of DNA is present. The C, values observed for theseunblocked isolates ranged from 22 to 30. This suggests that the amountof genomic DNA present at the start of the reaction differed by as muchas 256-fold from preparation to preparation.

Blocking is demonstrated by an increased C_(t) value for the detectionof the orfX subsequence, when blocker is present, as compared the C_(t)observed in the absence of the blocker polynucleotide. This value, thedelta C_(t) (C_(t) from PCR with blocker present—C_(t) from PCR withoutblocker) indicates the effectiveness of the blocker polynucleotideagainst a given strain. However, the delta C_(t) cannot be viewed as anabsolute value because the quantity of starting material variedsignificantly from reaction to reaction.

For example, a lysate that has a C_(t) of 30 in the absence of blockerand a C_(t) of 45 in the presence of blocker, has a delta C_(t) of 15.In this example a delta C_(t) of 15 indicates complete suppression ofthe amplification of the mg subsequence because a Ct of 45 indicates theno amplification product was detect in 45 PCR cycles. If the starting Ctis 20 and the delta Ct is 15 this would not represent a completesuppression of the orfX signal but rather ˜33,000-fold reduction.

The impact of the blocker polynucleotide was demonstrated in all of theisolates tested even though the efficency of blocker showed considerablevariation across the 80 isolates. In the presence of the blockerpolynucleotide, 35% of the isolates showed complete inhibition of theorfX amplification, 22% showed a 1000- to 16,000-fold reduction in orfXamplification, 15% showed a 64- to 1000-fold reduction in the orfXamplification and 28% showed an 8- to 64-fold reduction in the orfXamplification.

Published sequences of the orfX and attB regions of the S. aureus genomeshow that sequence polymorphisms exist between strains. Therefore, it isunlikely that a single blocker polynucleotide will be effective for allMSSA strains. Multiple blocker polynucleotides will be required tosuppress the amplification of target sequences within orfX regionspresent in the range of the MSSA strains encountered. Therefore, awell-designed blocker polynucleotide is expected to have a spectrum ofeffectiveness against a range of existing strains, effectively blockingthe amplification of orfX target sequences in a particular subset ofstrains with less complete suppression of orfX target sequences fromother strains. Therefore, in certain cases, it will be useful to includea “cocktail” of two or more blockers to achieve blocking over a broadspectrum of polymorphisms in the target polynucleotide sequenceassociated with different S. aureus strains.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed:
 1. A method for determining the presence or absence ofan insertion polynucleotide at a junction site in a targetpolynucleotide sequence, wherein the junction site in the targetpolynucleotide sequence is spanned by a first target sequence on oneside and a second target sequence on the other side, the first andsecond target sequences being contiguous when the insertionpolynucleotide is absent, the method comprising: contacting the targetpolynucleotide sequence with a first primer, a second primer, a blockerpolynucleotide, a polymerase, and one or more nucleotide triphosphatesand performing an amplification reaction, wherein: (i) the blockerpolynucleotide hybridizes to both contiguous first and second targetsequences when the insertion polynucleotide is absent, (ii) the firstprimer hybridizes to a first region of the first target sequenceindependent of the presence of the insertion polynucleotide, the firstregion being proximal to the junction site, and (iii) the second primerhybridizes to a second region of the first target sequence, the secondregion being distal to the junction site, wherein the second primer iscapable of priming synthesis of a portion of the first target sequencespanned by the first and second regions of the first target sequence,such that when the insertion polynucleotide is present at the junctionsite, the first and second primers support exponential amplification ofthe portion of the first target sequence spanned by the first and secondregions of the first target sequence, and when the insertionpolynucleotide is absent from the junction site, the blockerpolynucleotide hybridizes to the contiguous first and second targetsequences to block hybridization of the first primer to the first regionso that amplification of the portion of the first target sequencespanned by the first and second regions of the first target sequence isinhibited, whereby the presence or absence of the insertionpolynucleotide at the junction site in the target polynucleotidesequence is determined by the presence or absence of amplified portionof the first target sequence spanned by the first and second regions. 2.The method of claim 1, wherein the full length of the first primerhybridizes to the first region of the first target sequencecompetitively with the blocker polynucleotide.
 3. The method of claim 1,wherein a portion of the first primer hybridizes to the first region ofthe first target sequence competitively with the blocker polynucleotide.4. The method of claim 1, wherein first primer and the blockerpolynucleotide each is substantially complementary to the first regionof the first target sequence within the target polynucleotide sequenceand the first primer has fewer mismatched nucleotides than the blockerpolynucleotide relative to the first region of the first target sequencewithin the target polynucleotide sequence.
 5. The method of claim 1,wherein the first primer is completely complementary to the first regionof the first target sequence within the target polynucleotide sequenceand the blocker polynucleotide carries at least one internal mismatchcompared to the first region of the first target sequence within thetarget polynucleotide sequence.
 6. The method of claim 1, wherein theblocker polynucleotide comprises a moiety at its 3′-end selected fromthe group consisting of phosphate and hexylamine.
 7. The method of claim1, wherein the blocker polynucleotide comprises at least one nucleicacid analog base.
 8. The method of claim 1, wherein the nucleotidetriphosphates are dNTPs.
 9. The method of claim 1, wherein thepolymerase is a DNA polymerase.
 10. The method of claim 9, wherein theDNA polymerase is a Taq DNA polymerase.
 11. The method of claim 1,wherein the method is performed in a multiplex format.
 12. The method ofclaim 1, wherein amplification comprises polymerase chain reaction(PCR).
 13. The method of claim 1, wherein amplification comprises realtime (PCR).
 14. The method of claim 1, wherein the insertionpolynucleotide is at least 10 nucleotide bases in length.